1. Field of the Invention
The present invention relates generally to the field of protein engineering. More particularly, it concerns improved compositions of Fc antibody domains conferring high binding to FcγRIIB and altered effector function.
2. Description of Related Art
Currently, the top 25 marketed recombinant therapeutic antibodies have sales of well over $43.5 billion/year, and with a forecasted annual growth rate of 9.2% from 2010 to 2015, they are projected to increase to $62.7 billion/year by 2015 (J. G. Elvin et al., 2013). Monoclonal antibodies (mAbs) comprise the majority of recombinant proteins currently in the clinic, with 1064 products undergoing company-sponsored clinical trials in the USA or EU, of which 164 are phase III (Elvin et al., 2013). In terms of therapeutic focus, the mAb market is heavily focused on oncology and inflammatory disorders, and products within these therapeutic areas are set to continue to be the key growth drivers over the forecast period. As a group, genetically engineered mAbs generally have a higher probability of FDA approval success than small-molecule drugs. At least 50 biotechnology companies and all major pharmaceutical companies have active antibody discovery programs in place. The original method for isolation and production of mAbs was first reported at 1975 by Milstein and Kohler (Kohler and Milstein, 1975), and it involved the fusion of mouse lymphocyte and myeloma cells, yielding mouse hybridomas. Therapeutic murine mAbs entered clinical study in the early 1980s; however, problems with lack of efficacy and rapid clearance due to patients' production of human anti-mouse antibodies (HAMA) became apparent. These issues, as well as the time and cost consumption related to the technology, became driving forces for the evolution of mAb production technology. Polymerase Chain Reaction (PCR) facilitated the cloning of monoclonal antibody genes directly from lymphocytes of immunized animals and the expression of combinatorial libraries of antibody fragments in bacteria (Orlandi et al., 1989). Later libraries were created entirely by in vitro cloning techniques using naive genes with rearranged complementarity determining region 3 (CDR3) (Griffths and Duncan, 1998; Hoogenboom et al., 1998). As a result, the isolation of antibody fragments with the desired specificity was no longer dependent on the immunogenicity of the corresponding antigen. These advantages have facilitated the development of antibody fragments to a number of unique antigens including small molecular compounds (haptens) (Hoogenboom and Winter, 1992), molecular complexes (Chames et al., 2000), unstable compounds (Kjaer et al., 1998), and cell surface proteins (Desai et al., 1998).
One method for screening large combinatorial libraries of antibodies to identify clones that bind to a ligand with desired affinity involves expression and display of antibody fragments or full length antibodies on the surface of bacterial cells and more specifically E. coli. Cells displaying antibodies or antibody fragments are incubated with a solution of fluorescently labeled ligand and those cells that bind said ligand by virtue of the displayed antibody on their surface are isolated by flow cytometry. In particular, Anchored Periplasmic Expression (APEx) is based on anchoring the antibody fragment on the periplasmic face of the inner membrane of E. coli followed by disruption of the outer membrane, incubation with fluorescently-labeled target, and sorting of the spheroplasts (U.S. Pat. No. 7,094,571, Harvey et al., 2004; Harvey et al., 2006).
The receptors for Fc domain of antibodies are expressed on diverse immune cells and are important in both promoting and regulating the immunological response to antibody antigen complexes (called immune complexes). The binding of the Fc region of antibodies that have formed immune complexes with a pathogenic target cell to different Fc receptors expressed on the surface of leukocytes to elicit antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) or complement-mediated reactions including complement dependent cytotoxicity (CDC).
In humans there are two general classes of FcγRs for IgG class antibodies: activating receptors, characterized by the presence of a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) sequence associated with the receptor, and the inhibitory receptor, characterized by the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence (Daeron M, 1997 and Bolland S et al., 1999). Of note, activating FcγRs, FcγRI, FcγRIIA, FcγRIIIA, FcγRIIIB induce activating or pro-inflammatory responses, while inhibitory FcγRIIB induces anti-inflammatory or inhibitory responses. Among activating FcγRs, FcγRIIA and FcγRIIIA have natural allotypes which can affect binding capacity of IgG. FcγRIIAH131 showed higher binding affinity than FcγRIIAR131 for IgG and FcγRIIIAV158 showed higher binding affinity than FcγRIIIAF158 for IgG. All naturally produced antibodies and also recombinant glycosylated antibodies produced by tissue culture contain Fc domains that bind to both the activating and the inhibitory FcγRs. (Boruchov et al. 2005; Kalergis et al., 2002).
As mentioned above, aglycosylated antibodies do not display any detectable binding to FcγRIIB. Due to the physiological importance of Fc binding to FcγRIIB and the importance of Fc binding to FcγRIIB with therapeutic antibodies (e.g., agonistic antibodies), there is a clear need for new Fc domains, and in particular aglycosylated Fc domains, that can selectively bind FcγRIIB.